Bactericidal/permeability-increasing protein is a 50 to 60 kilodalton (kDa) protein, isolated from the granules of mammalian polymorphonuclear leukocytes (hereafter "PMN"), blood cells that are essential in the immune defense against invading microorganisms in mammals. BPI is known to occur only in the myeloid series of blood cells, is produced at the promyelocytic/myelocytic stage of differentiation and is located in the primary granules in these cells.
BPI is a potent bactericidal agent active against a wide range of gram-negative bacterial species. It is highly effective and specific as a cytotoxic agent, i.e., 1-10 nM (0.05-0.5 micrograms) kills more than 90% of a population of 10.sup.7 sensitive (i.e. gram-negative) bacteria. 100-fold higher concentrations of BPI are non-toxic to other microorganisms or eukaryotic cells. All available evidence suggests that in the intact PMN as well as in crude leukocyte fractions, BPI is the principal oxygen-independent bactericidal agent active against gram-negative bacteria.
BPI isolated from both human and rabbit PMN has been purified to homogeneity. The molecular weight of human BPI is approximately 58,000 Daltons (58 kDa) and that of rabbit BPI is approximately 50 kDa. These two proteins have a similar amino acid composition and the amino acid sequence of the first 15 NH.sub.2 -terminal amino acid residues is also similar in rabbit and human BPI. Both proteins are highly basic, having an isoelectric point greater than 9.6. The entire amino acid sequence of human BPI has also been elucidated (as disclosed in copending commonly assigned U.S. patent application Ser. No. 228,035, filed Aug. 5, 1988, along with cDNA sequences encoding the BPI protein and cells transfected with the cDNA sequences).
The biological effects of BPI require attachment of the protein to the surface of susceptible gram-negative bacteria. Initial binding of BPI to target cells involves electrostatic interactions between the basic protein and the negatively charged sites on the lipopolysaccharides (LPS) in the bacterial outer membrane leading to an activation of bacterial enzymes that degrade phospholipids and peptidoglycans. The final stage of action is the actual killing of the bacteria by an as yet unknown mechanism. The closely similar amino acid composition and nearly identical bactericidal and membrane-perturbing properties of BPI purified from human and rabbit PMN suggest that this protein has been highly conserved during evolution and is an important member of the anti-bacterial arsenal of the mammalian PMN.
U.S. patent application Ser. No. 084,335 filed Aug. 6, 1987, now abandoned (in favor of its C-I-P, Ser. No. 228,035), discloses biologically active fragments of human and rabbit BPI proteins and methods to produce and use these proteins to combat infections caused by gram-negative bacteria. The fragments were approximately 25 kDa molecular weight and contained all of the biological activities of the entire protein.
Due to its potent bactericidal action against gram-negative bacteria and lack of cytotoxicity towards other microorganisms and eukaryotic cells, it is anticipated that BPI may be employed as a chemotherapeutic agent and/or as a model for the design of new antibiotic agents. It would be highly useful, therefore, to find an agent which potentiates or enhances the effectiveness of BPI against gram-negative bacteria, alone or in combination with other chemotherapeutic agents and/or antibiotics in killing gram-negative bacteria thereby allowing the use of lower effective amounts of BPI.